AN ABSTRACT OF THE THESIS OF Robert H. Kaiser for the Master of Science in presented on August 4, 1978 Title: The Effect of Inorganic Phosphate on the Production of Non-volatile, Aliphatic Organic Acids by Aspergillus wentii Abs tract approved: 6J ckJff .... >.' ' g ... '\ _ Inorganic phosphate has long been known to affect organic acid production and the types of acids produced are related to its concentration. In this study the effect of the variation of inorganic phosphate concentration on the production of non-volatile, aliphatic organic acids by a fungus was investigated. To find a suitable test organism 200 soil samples were taken and 306 acid producing specimens were isolated from them. Aspergillus wentii from these isolates was selected because of its ability to produce large amounts of several organic acids and its cultural characteristics. In A. wentii cultures having low phosphate concentrations (0.025 and 0.05 g phosphate/l) citric acid production was initiated first, while in the higher phosphate concentrations (0.5 and 1.0 g phosphate/l) malic acid was
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AN ABSTRACT OF THE THESIS OF
Robert H. Kaiser for the Master of Science
in Biolo~ presented on August 4, 1978
Title: The Effect of Inorganic Phosphate on the Production
of Non-volatile, Aliphatic Organic Acids by Aspergillus
Figure 6. Dry weight, pH and Relative Acid Concentration in the 1.0 g phosphate/l culture.
20
~rs and was not seen before that • i:<··,
time. Its hi9h~~~~~~on indicates that it was being
very actively pr~,~tthat time.
The affec~;.':Q'f,the variation of the phosphate concen
tration on the initiation of organic acid synthesis is the
opposite of that reported by S. M. Martin and R. Steel (1955)
in their work with Aspergillus niger. In their study the
addition of phosphate caused citric acid to be produced
before malic acid. In this investigation ~. wentii produced
malic acid first when phosphate was added. It would appear
that low phosphate concentrations facilitate the start of
the production of citric acid, while the higher phosphate
concentration cause the initiation of malic acid. However,
the effects Martin and Steel reported "appeared to be true
phosphate effects rather than pH effects, since the addition
of phosphate did not alter the shape of the pH curves or the
final pH (Martin and Steel, 1955)." In this study the same
can be said as the pH changes were rapid, they did not
correlate with the other activities that were measured, and
the final pH of the cultures were not much different.
Other experiments were attempted with phosphate
concentrations up to 30 gil, but were not reported here.
Changes in pH indicated that acid was being produced in the
cultures, but increasing the phosphate concentration beyond
3 to 5 gil resulted in phosphate ion interference that made
the TLC assay impossible using the Denison and Phares (1952)
21
solvent system. The acids could not be separated. Further
more, the capacity of the ion exchange resin (in the amount
of resin used in this study) was over reached with the high
level of anion. However, further study of these higher
phosphate concentrations using the solvent system developed
by this investigator might be able to separate the acids,
thus allowing their identification. Increasing the amount
of ion exchange resin in the columns would also help because
of the high amount of phosphate anion.
In the higher phosphate study when phosphate concen
trations were increased above 15 gil, growth started to be
inhibited in the cultures until at 30 gil no growth occurred
in the culture medium. These results along with those from
the low phosphate cultures show that the phosphate concen
trations between 0.05 g and 15 gil permit maximum growth
which was predicted by Weinberg (1974).
Other effects unrelated to phosphate concentration
were observed. Malic acid and citric acid are produced in
excess during the balanced growth phase. Therefore, they
are unusual cases of metabolism. A few reports of secondary
metabolites being produced during balanced growth have been
reviewed by Bullock (1975). In the work of Martin and Steel
(1955) citric acid and malic acid accumulated during the
balanced growth phase although the concentration of citric
acid greatly increased after balanced growth ended and malic
acid remained at low levels. The decrease or disappearance
22
of malic acid from the medium after balanced growth ends
conflicts with the generally accepted definitions of
secondary metabolism as stated before. Thus, malic acid in
Aspergillus wentii must be considered a special case with
reference to secondary metabolism. It is the opinion of this
investigator that malic acid is a secondary metabolite, since
it does meet most of the criteria of a secondary metabolite
as defined by Bullock (1975).
Any attempt to explain the disappearance of the malic
acid from the medium would require the consideration of
several interconnected factors. One reason may be related
to the accumulation of citric acid and fumaric acid. During
cellular regulation of the citric acid cycle (Lehninger,
1975), citric acid might become more concentrated if enzymes
that produce it are more active or more abundant or its pre
cursors are processed more efficiently. If this be the case,
malic acid would be processed to oxalacetic acid more readily.
Fumaric acid accumulation might mean that the enzymes
catalyzing the synthesis of malic acid are limited in some
way or that the enzymes producing fumaric acid are more ac
tive or abundant. Because fumaric acid appears late in the
fermentation it is a typical example of a secondary metab
olite as defined before.
In a consideration of the unexpected deceleration in
the growth rate following the balanced growth phase and just
prior to the storage phase, one may postulate that a critical
23
nutrient has been exhausted, thus arresting growth (Bullock,
1975). With the end of replication new enzymes are produced
for the synthesis of storage products, but do not add much
weight to the mycelium. This would explain the deceleration,
since little additional mass can be added to the cell until
the new enzymes are synthesized.
As to what caused the end of balanced growth in the
cUltures, the results indicate that the exhaustion of phos
phate in the cultures having low phosphate levels ended
balanced growth and that the exhaustion of some other
nutrient, perhaps nitrogen, arrested it in the high phosphate
cultures.
Since all these activities--citric and fumaric acid
accumulation, the unusual production and decrease of malic
acid, nutrient exhaustion and the deceleration in mass
accumulation--are associated with the period of time when
balanced growth slows, it would seem that A. wentii has the
same traits as other organism when it produces secondary
metabolites. Although it grows somewhat slower than other
organisms (Martin and Steel, 1955), its pattern of secondary
metabolism is essentially the same as other filamentous
fungi as described by Bu'lock (1975).
In the further study of the growth and acid produc
tion of A. wentii several areas need more investigation. A
method needs to be developed to quantitate the acids produced
such as the uv technique developed by Richards (1975).
Replication growth could be better monitored by
assay for the nucleic acids or by using one
suggested by Bullock (1975) rather than dry weight
has limitations when used with the fungi.
of the organism could be examined by using assays for
carbohydrates and lipids. Finally, a longer fermentation
period covering a wider range of phosphate concentrations is
also needed.
In summary, this study has shown that phosphate does
affect the organic acid production of A. wentii. In addition,
it has given some knowledge of the growth characteristics of
the organism on synthetic media.
BIBLIOGRAPHY
Berry, D. R. 1975. The Environmental Control of the Physiology of Filamentous Fungi. pp. 16-32. In Smith, J. E. and D. R. Berry (eds.), The Filamentous Fungi: Vol. 1. Industrial Mycology. Halstead Press, New York.
Borrow, A., E. G. Jefferys, R. H. J. Kessell, E. C. Lloyd, P. B. Lloyd, and I. S. Nixon. 1961. The Metabolism of Gibberella fujikuroi in stirred culture. Canadian Journal of Microbiology. 7:227-276.
Bu'lock, J. D. 1975. Secondary Metabolism in Fungi and Its Relationship to Growth and Development. pp. 33-56. In Smith, J. E. and D. R. Berry (eds.), The Filamentous Fungi: Vol. 1. Industrial Mycology. Halstead Press, New York.
Currie, J. N. 1917. The Citric Acid Fermentation of Aspergillus niger. Journal of Biological Chemistry. 31:15-37.
Demain, A. L. 1968. Regulatory Mechanisms and the Industrial Production of Microbial Metabolities. Lloydia. 31:395-418.
Denison, F. W. and E. F. Phares. 1952. Rapid Method for Paper Chromatography of Organic Acids. Analytical Chemistry. 24:1628-1629.
Dyson, R. D. 1974. Cell Biology: A Molecular Approach. Allyn and Bacon, Inc., Boston.
Foster, J. W. 1949. Chemical Activities of Fungi. Academic Press, New York.
Karow, E. o. and S. A. Waksman. 1947. Production of Citric Acid in Submerged Culture. Industrial and Engineering Chemistry. 39:821-825.
Lilly, V. G. and H. L. Barnett. 1951. Physiology of the Fungi. McGraw-Hill, New York.
Lehninger, A. L. 1975. Biochemistry: The Molecular Ba$~S of Cell Structure and Function. Worth Publ., New Y~l.
25
26
Martin, S. M. and R. Stell. 1955. Effect of Phosphate on Production of Organic Acids by Aspergillus niger. Canadian Journal of Microbiology. 1:470-472.
Miall, L. M. 1975. Historical Development of Fungal Fermentation Industry. pp. 104-121. In Smith, J. E. and D. R. Berry (eds.), The Filamentous Fungi: Vol. 1. Industrial Mycology. Halstead Press, New York.
Raper, K. B. and K. I. Fennell. 1973. The Genus Aspergillus. Krieger Publ. Co., New York.
Richards, M. 1975. Separation of Mono- and Dicarboxylic Acids by Liquid Chromatography. Journal of Chromatography. 115:259-261.
Smith, J. E. and D. R. Berry. 1974. An Introduction to Biochemistry of Fungal Development. Academic Press, New York.
Shu, P. and M. J. Johnson. 1948. Citric Acid Production by Submerged Fermentation with Aspergillus niger. Industrial and Engineering Chemistry. 40:1202-1205.
VanEtten, C. H. and C. E. McGrew. 1957. Ion Exchange Micromethods for Separation of Fermentation Acids. Analytical Chemistry. 29:1506-1509.
Vezina, C. and K. Singh. 1975. Transformation of Organic Compounds by Fungal Spores. pp. 158-192. In Smith, J. E. and D. R. Berry (eds.), The Filamentous Fungi, Vol. 1. Industrial Mycology. Halstead Press, New York.
Weinberg, E. D. 1974. Secondary Metabolism: Control by Temperature and Inorganic Phosphate. Developments in Industrial Microbiology. 15:70-81.